Method for preparing agonist for improving boar sperm motility

A method for preparing an agonist for improving boar sperm motility, comprising:

• (1) collecting a testicular tissue from a young boar 3 days after born, and infiltrating the testicular tissue in normal saline containing 1% of a double-antibody at 37° C. for 10-30 min;
• (2) washing the infiltrated testicular tissue three times with PBS containing 1% of the double-antibody;
• (3) incubating the testicular tissue in DMEM/F12 media at 37° C. for 30 min to fully disperse the testicular tissue;
• and transferring the dispersed testicular tissue to a centrifuge tube followed by centrifugation to remove a supernatant;
• (4) adding 1.5 mL of hyaluronidase and 1.5 mL of collagenase IV to the centrifuge tube;
• shaking the centrifuge tube violently at 37° C. every other 2 min;
• after continuously shaken 2-3 times, adding 3 mL of DMEM/F12 media;
• and centrifuging the centrifuge tube to remove a supernatant;
• (5) adding 1.5 mL of trypsin and 1.5 mL of deoxyribonuclease to the centrifuge tube;
• shaking the centrifuge tube violently at 37° C. every other 2 min;
• after continuously shaken 2-3 times, adding 3 mL of DMEM/F12 media to the centrifuge tube to produce a first cell suspension;
• (6) filtering the first cell suspension sequentially by 80-mesh, 200-mesh and 300-mesh cell sieves;
• (7) centrifuging the filtered first cell suspension to remove a supernatant;
• adding 4-6 mL of a cell culture medium to form a second cell suspension;
• mixing the second cell suspension by blowing;
• transferring the second cell suspension to a cell culture dish to culture for 2 h;
• and replacing the cell culture medium;
• (8) passaging the cells cultured in the cell culture dish;
• and inoculating 1×106 cells to each of cell culture dishes having a diameter of 6 cm;
• and
• (9) taking out the cell suspensions in the cell culture dishes after cultured for 8 h, and cryopreserving the cell suspensions at −20° C.